COMPARISON OF XENOBIOTIC METABOLIZING ENZYME ACTIVITIES IN NORMAL HUMAN SKINAND RECONSTRUCTED HUMAN SKIN MODELS FROM SKINETHIC LABORATORIES

J. Eilstein1 • G. Léreaux1 • J.R. Meunier1 • J. Leclaire1 • D. Duché1

1L’Oréal Research Life Sciences Department, 93601 Aulnay-Sous-Bois, Cedex, France.Corresponding author: jeilstein@rd.loreal.com.

Skin stands for the major protective barrier of the body to its environment. Also, skin is an organ involved in the metabolism of xenobiotics and its ability to metabolize them can become consequent when considering its total surface area (2 square meters). Consequently, research on skin metabolism would need a real scientific effort to characterize skin metabolizing enzymes and their activities. In addition, the 7th European amendment to the cosmetic directive forbids the use of animal testing to assess the efficacy and safety of new cosmetic ingredients. This policy has forced the cosmetic industry to develop in vitro tools such as reconstructed human skin models (skin models) as alternative methods to animal experiments. For these reasons, these skin models require to be characterized and compared with normal human skin (NHS) samples in terms of metabolic capabilities. mRNA expression of several enzymes (CYP450, Esterase, ADH, ALDH, NAT, GST, UGT, SULT…) were previously demonstrated. This work presents their apparent catalytic parameters determination (apparent Km, Vmax and the ratio Vmax/Km) in skin models compared with NHS. Results show that all these enzymes involved in the metabolism of xenobiotics are expressed and functional in the NHS and skin models. Also, the Vmax/Km ratios(estimating the intrinsic metabolic clearances) show that the metabolic abilities are the most often comparable between the skin models and NHS. These results indicate that the skin models can substitute themselves for NHS to select cosmetic ingredients on the basis of their metabolism, efficacy or/and safety.

ABSTRACT

INTRODUCTION RESULTSMATERIALS AND METHODS

1. mRNA

Expression profile of phase 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models EpiskinTM and full thikness model from EpiskinTM

Van Luu-The, Daniel Duché, Corinne Ferraris, Jean-Roch Meunier, Jacques Leclaire, Fernand LabrieJ Steroid Biochem Mol Biol. 2009 Sep;116(3-5):178-86

2. RECONSTRUCTED HUMAN SKIN MODELS

1. LIVER & SKIN

Hepatocytes

Endogenous & Drug metabolisms

2% body weight, 1.5 kg

Keratinocytes

Barrier function , thermoregulation...

15% body weight , 2 m²

Main cell type

Main functions

Weight

➡ CHARACTERIZATION OF THE SKIN METABOLIC CAPABILITIES

1. DEVELOPPED APPROACH

Protein expression(immuno histochemistry)

(Western blots)Gene expression(RT-PCR)

Enzyme activityRadio, MSn, fluo or UV-HPLC

2. REGULATORY CONTEXT

➡ USE OF RECONSTRUCTED HUMAN SKIN MODELS

➡ COMPARISON OF THE METABOLIC CAPABILITIES OF SKIN MODELS TO NORMAL HUMAN SKIN

2. RECONSTRUCTED HUMAN SKIN MODELS

Phase I Metabolizing Enzyme Activities1

Phase II Metabolizing Enzyme Activities2

➡ NORMAL HUMAN SKIN VS SKIN MODELS: KMAPP AND VMAXAPP ARE DIFFERENT BUT VMAX/KM RATIO ARE EQUIVALENT

CYP450 Activities (n=4) ES Activity (n=4) ADH (n=4) ALDH (n=4)

GST Activity (n=4) NAT1 Activity (n=4) UGT Activity (n=4) SULT Activity (n=4)

DISCUSSION AND CONCLUSION

➡ SKIN CAN BE INVOLVED IN DIFFERENT METABOLIC PROCESSES (potential First Pass Effect)

➡ SKIN IS RATHER AN ORGAN OF DETOXIFICATION THAN A BIO ACTIVATING ONE

➡ SKIN TOXICITY APPEARS WHEN: - DETOXIFICATION SYSTEMS ARE OVER EXPOSED TO TOXICANTS - REACTIVE MOLECULES ARE RELEASED IN LARGE AMOUNTS

➡ RECONSTRUCTED HUMAN SKIN MODELS ARE GOOD ENOUGH PREDICTIVE OF WHAT IT CAN OCCUR AT THE IN VIVO SKIN LEVEL

➡ DEDICATED TOOLS FOR SKIN METABOLISM AND TOXICITY STUDIES

2. COMPARISON OF METABOLIZING ENZYME ACTIVITIESON THE BASIS OF THE APPARENT INTRINSEC METABOLIC CLEARANCES (Vmax/Km RATIOS)1. ENZYME ACTIVITIES

MODEL COMPARISON PER ACTIVITY:

➡ NHS metabolic clearances are highly variables

➡ Model metabolic clearances are often similar to NHS clearances except for ESEPIS&RHE, ADHEPIS, NATFTM

Results expressed as the Mean ± sem in µL.mg protein-1.min-1

• Low basal expression and activity of CYP450 involved in «Drug metabolism» (!! induction !!)• High esterase activity (Low affinity with the compound used as substrate…) • ADHandALDHdetectedandtheirrespectiveactivitiesquantified

• NATactivitydetectedandquantified• GSTactivitydetectedandquantified• UGTactivitydetectedandquantified• VerylowbasalSULTactivityexceptforsulfationofsteroidsuchaDHEA or cholesterol

• Otherenzymestobequicklytested: -PhaseI:Peroxidases -PhaseII:Catecholorthomethyltransferase(COMT)

Normal Human Skin (NHS):Epidermis/Dermis

(BIOPREDIC)

EpiskinTM:Reconstructed human epidermis

SkinEthicTM RHE:Reconstructed human epidermis

Full thickness model of EpiskinTM:Reconstructed human epidermis/

equivalent dermis

•Breastreductions

•NHK(Breastsurgery)•Pool4-5donors Support: BPER

•NHK(foreskin/abdomen)•1donor/Pool2donors Support: Polycarbonate

•NHK(Breastsurgery)•Pool4-5donors Support: Polycarbonate

IN MOST OF CASES, MODELS ARE SIMILAR TO NHS IN TERMS OF METABOLIC CAPABILITIES

a incubation with α−naphtoflavone as inhibitorc incubation with azamulin as inhibitor